Uncovering the mechanisms which regulate the expression of genes during the development of multicellular organisms will be essential for our understanding of developmental biology as a whole. This knowledge will also be useful for understanding and treating "developmental diseases" such as cancer and birth defects. The Drosophila dopa decarboxylase gene is regulated in a tissue and developmental stage specific manner. The enzyme encoded by this locus is involved with the hardening of the exoskeleton or cuticle after molting and in neurotransmitter biosynthesis. High levels of enzyme activity are found only during cuticle hardening and only in the tissue that gives rise to that cuticle. Cloned copies of the wildtype Ddc gene have already been demonstrated to function properly after being reintegrated into the genome via the recently developed Drosophila DNA transformation system. These reintegrated genes are regulated properly. The proposed experiments will use in vitro mutagenesis to identify and analyze the control regions of a cloned copy of the gene. The phenotypes of these mutations will be assayed in vivo after reintegration. These mutations, ranging from deletions to single base pair substitutions, will define both the structure and the function of the individual regulatory sites. These findings will be extended by combining the in vitro constructed mutants with in vivo genetics in the selection of second site mutations which suppress specific Ddc control mutations. Mutations mapping outside of Ddc will identify the genes which encode the trans-acting factors that regulate Ddc expression. This will set the stage for future studies of the physical interactions between the cis- and trans-acting controls and possibly studies of the coordinate regulation of groups of genes during development.